This project is submitted under Program Announcement (PA) Number: PAR-12-060 and Title: Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes (R01). Opioid receptors are well known to be intimately involved with modulating the perception of pain. Throughout history, opioid preparations have been utilized to effectively manage pain and have continued to be the mainstay of therapy due to their efficacy. However, these drugs are associated with the liabilities of dose-limiting side-effects, tolerance and addiction. Still they are the most widely prescribed medications to treat acute and chronic severe pain. The search for an effective analgesic compound devoid of side-effects, is still a large goal of the medical establishment. Only recently in history was it realizd that endogenous opioid-modulating peptides exist that can be utilized as a means of additional therapy to attenuate these liabilities. These opioid modulating peptides (specifically cholecystokinin and Neuropeptide FF) are able to both reduce and potentiate the pharmacological effects of opioids depending on the dose and site of action. More interestingly, they have been proposed to have a role and possible responsibility for tolerance and dependence associated with opioid therapy. Specifically, Neuropeptide FF (NPFF) has been reported to exist as an endogenous anti-opioid system and block the development of tolerance and opioid-induced hyperalgesia. There are two recognized receptors in this system, NPFF1 and NPFF2. Currently, established (subtype selective or non-selective) small molecule, non-peptide probes for the NPFF system do not exist. This lack of a suitable non-peptide probe has hampered the full understanding of the actions of NPFF and how this receptor system is involved in the actions of opioids as well as other proposed pharmacologies. It is the goal of this project to identify one or more suitable non-peptide probes to serve the research community in an effort to further characterize the NPFF system. Our lead molecules, generated under our project R03 DA029738, show that we have both angonists and antagonists for this system and have begun to understand selectivity issues yet they need to be optimized for in vivo approaches. This work will be carried out through the following specific aims: AIM 1 - To prepare through chemical synthesis, novel non-peptidic NPFF1 and NPFF2 ligands, agonists or antagonists with selectivity for NPFF1, NPFF2, or a combination of affinities at each receptor; AIM 2 - To investigate the physiochemical properties of high affinity NPFF ligands through a variety of in vitro assays that will assess solubility, stability, membrane permeability, protein binding, microsomal stability and metabolic profiling; AIM 3 - To perform in vivo pharmacokinetics in rats to determine half-life, bioavailability and brain to plasma ratios. Those compounds that show activity will be subjected to full PK workup in rats to ultimately determine their suitability as potential in vivo probes and to understand their dosing requirements (peak time, brain:plasma, half-life, clearance); AIM 4 - To evaluate novel NPFF ligands in vivo for hyperalgesic activity. Compounds will be evaluated in a warm-water tail-withdrawal hyperalgesia model to confirm and further determine pharmacological profiles of those compounds identified in Aims 2 and 3. Overall, with our expertise through this project, we will provide the research community with suitable in vivo non-peptide probes to further characterize the NPFF system. These will be new tools as currently, non-peptide NPFF ligands with favorable affinities, activities, and in vivo properties do not exist.